JP6275668B2 - Nanoparticle-mediated delivery of sequence-specific nucleases - Google Patents

Description

(Cross-reference of related applications)
This application claims the benefit of US Provisional Patent Application No. 61 / 167,389 (filed Apr. 7, 2009).

The unique properties of nanoparticles can be exploited to deliver DNA into cells. Among the nanoparticles studied (eg, tungsten, aluminum, nickel, etc.), gold nanoparticles (GNP) tend to be excellent candidates for delivering DNA. Gold nanoparticles are preferentially selected for transformation due to their low cytotoxicity and ease of functionalization with various biologically important ligands. Gold nanoparticles range in size from 1.2 nm to 6
Up to 00 nm. The commonly used synthesis of GNP is negatively charged (eg,
For particles with a surface of 20 nm to 400 nm (citrate coated) the GNP in the smaller 1 nm to 10 nm range is positively charged. Plasmid DN
A is flexible enough to partially unravel its base, so that plasmid DNA can be exposed to gold nanoparticles. In the case of citrate functionalized GNP, the plasmid DNA can be partially lysed. The negative charge on the DNA backbone is well separated, so that the base and
The van der Waals attraction between the gold nanoparticles causes the plasmid DNA to bind and cover the surface of the gold particle. In contrast, in the case of positively charged GNPs, electrostatic forces and van der Waals forces can contribute to DNA coating or binding.

In addition to metal nanoparticles, semiconductor nanoparticles with a size range of 3 nm to 5 nm (e.g.,
Quantum dots) (“QD”) have also been used as carriers for delivering various molecules into cells. DNA and proteins can be coated or linked to a QD surface that is polyfunctionalized with a ligand (eg Patolsky, F. et al., J. Am. Chem. Soc., 125, 13918 (2
003)). QDs polyfunctionalized with carboxylic acids or amines can be cross-linked to molecules containing thiol groups (see eg Dubertret B. et al., Science, 298, 1759 (2002).
); Akerman, ME, WCWChan, P. Laakkonen, SNBhatia, E. Ruoslahti, Proc. Natl. A
cad. Sci. USA, 99, 12617 (2002); Mitchell, GP, CAMirkin, RLLetsinger, J.
Am. Chem. Soc., 121,8122 (1999)) or a molecule containing an N-hydroxysuccinimidyl (NHS) ester group by using standard bioconjugation protocols (Eg Pinaud, F., D. King, H.-P. Moore,
S. Weiss, J. Am. Chem. Soc., 126, 6115 (2004); Bruchez, M., M. Moronne, P. Gin, S. Weis
s, APAlivisatos, Science, 281, 2013 (1998)). An alternative is to polyfunctionalize QD by conjugation with streptavidin. Streptavidin forms conjugates with biotinylated proteins, oligos or antibodies (eg Dahan, M. et al., Science, 302, 442 (2003); Pinaud, F., D. King, H.- P. Moore, S. Weis
s, J. Am. Chem. Soc., 126, 6115 (2004); Dahan, M. et al., Science, 302, 442 (2003); Wu.XY
Nature Biotechnol., 21, 41 (2003); Jaiswal, JK, H. Mattoussi, JMMauro, SMS
imon, Nature Biotechnol., 21, 47 (2003); and Mansson, A. et al., Biochem. Biophys. Res.
Commun., 314, 529 (2004)).

Nanoparticles have been used to deliver plasmid DNA to various animal cells. D
It has been found that when NA-coated nanoparticles are incubated with cells that do not have cell walls, the cells take up the DNA-coated nanoparticles and begin to express any gene encoded by the DNA. When nanoparticle delivery to cells that normally have a cell wall is desired, the cell wall is removed before the particles are added to the protoplast (Torney, F. et al.,
Nature Nanotechnol. 2, (2007)). In plant cells, the cell wall acts as a barrier to the delivery of externally added molecules. Gene gun (biolistic method),
Many invasive methods such as microinjection, electroporation and Agrobacterium have been used to achieve gene and small molecule delivery into these plant cells surrounded by cell walls. Delivering small molecules and proteins across plant walls and into plant cells is advantageous for the development of enabling techniques for in vitro and in vivo manipulation of intact plant cells, tissues and organs It is thought that.

Although well established in bacterial cells, yeast cells, animal cells and moss, gene addition, ie the introduction of foreign DNA into a given genomic location, remains an important issue in higher plants. Even when the introduced DNA contains a large area of sequence homologous to the host DNA, site-specific transgene integration occurs at a much lower frequency in plant cells compared to random integration ( Halfter et al., 1992; Lee et al., 1990; Mia and Lam (1995)). For example, a highly efficient Agrobacterium-based transfection system and herbicide selection resulted in a gene targeting frequency of up to 5 × 10 ⁻⁴ in rice. Previous attempts to increase gene targeting efficiency in plants have included the use of negative selectable markers, and
Included is the use of plants that are genetically engineered to exhibit greater targeting frequencies. Despite these efforts, random DNA integration by non-homologous processes continues to be a major obstacle to gene targeting in plants. Given the general utility envisaged for targeted gene addition in the modification of crops for agricultural and industrial biotechnology, a solution to this problem is highly sought.

In this context, a substantial increase in the frequency of gene targeting in a wide variety of plant and animal model systems is associated with DNA double-strand breaks (DSBs) (DSBs) at specific genomic locations in the host cell. Has been observed after induction of innate cellular processes, ie, homology-directed DSB repair. A naturally occurring site-specific endonuclease whose recognition site is rare in the plant genome to allow transgene integration into a target sequence previously transferred into the plant genome by random integration Used in this style. While these studies emphasize the potential of targeted DSB introduction to stimulate gene targeting in plant cells, the challenge of introducing DSB into native loci remains.

In animal cells, solutions to targeted genome preparation / manipulation are achieved by various nucleotide sequence-specific binding proteins such as leucine zippers, zinc finger proteins, and the like. These proteins are involved in gene regulation as transcription factors,
And / or can be used to direct DSBs to native genomic locations. DS
B is provided by several different classes of sequence-specific nucleases (eg, meganucleases, etc.), leucine zippers, zinc finger proteins, etc., and more recently by the development of new chimeras of these proteins. obtain. One of the best described nucleotide-specific binding proteins is zinc finger protein (ZFP)
). C2H2 zinc fingers were found in the amphibian transcription factor TFIIIA and have since been found to be the most common DNA recognition motif in all species of metazoans. Depending on the X-ray crystal structure of Zif268 of C2H2 type ZFP, each zinc finger defines a 3 bp or 4 bp sub-site in a tandem configuration,
A remarkably segmental mode of protein-DNA recognition was revealed and the possibility of using this peptide motif as a scaffold for a DNA binding domain with unprecedented specificity was suggested. Since then, a large number of ZFPs engineered to bind novel sequences have been successfully used in many different laboratories in connection with artificial transcription factors and other functional chimeric proteins. The C2H2 zinc finger protein domain is
Used as a scaffold for sequence-specific DNA binding (Pavelitch and Pabo, 1991)
Various ZFNs have also been produced by fusing the zinc finger protein domain with a sequence-independent nuclease domain derived from the type IIS restriction endonuclease FokI (Kim et al., 1996). Engineered ZFNs are transformed into highly efficient targeting to endogenous genomic loci (Moehle et al., 2007) and primary human cells (Lomb
ardo et al., 2007).

Early attempts aimed at using ZFNs in plants remain promising (Lloyd et al., 2005; Wright et al., 2005; Maeder et al., 2008). A construct that retains the ZFN gene along with its corresponding recognition sequence under the control of an inducible promoter is stably integrated into Arabidopsis, which is targeted from non-homologous ends joining at the recognition site. It has been shown that introduced mutations are introduced with an average frequency of 7.9% in induced offspring seedlings (Lloyd et al., 2005). Similarly, out of 66 tobacco plants regenerated from protoplasts transformed with ZFN designed to cut at the SuRA locus, 3
Showed a single base pair deletion at the target site resulting from non-homologous end-joining repair (Maeder et al., 2008
). Tobacco cells containing a pre-integrated non-functional reporter gene that has lost 600 bp immediately adjacent to the zinc finger recognition sequence have a corresponding ZFN gene and a pre-integrated sequence that contains the 600 bp missing. Evidence for homology-directed repair of reporter genes when cotransformed with a construct containing donor DNA homologous to (Wright et al., 2005). Most recently, yeast-based assays have been used to identify ZFNs capable of cleaving plant endochitinase genes (Cai et al., 2009).
. Ti with both such a ZFN and a donor DNA construct containing a pat herbicide resistance gene cassette flanked by a short section with homology to the endochitinase locus
Agrobacterium delivery of the plasmid resulted in the incorporation of up to 10% targeted homology-directed transgene exactly into the ZFN cleavage site. It is important to note that other zinc finger designs based on the C3H1 design have been demonstrated in plants (Shukla et al., 2009; Cai et al., 2009).

Patolsky, F. et al., J. Am. Chem. Soc., 125, 13918 (2003) Dubertret B. et al., Science, 298, 1759 (2002) Akerman, M.E., W.C.W.Chan, P. Laakkonen, S.N.Bhatia, E. Ruoslahti, Proc. Natl. Acad. Sci. U.S.A., 99, 12617 (2002) Mitchell, G.P., C.A. Mirkin, R.L. Letsinger, J. Am. Chem. Soc., 121, 8122 (1999) Pinaud, F., D. King, H.-P. Moore, S. Weiss, J. Am. Chem. Soc., 126, 6115 (2004) Bruchez, M., M. Moronne, P. Gin, S. Weiss, A.P. Alivisatos, Science, 281, 2013 (1998) Dahan, M. et al., Science, 302, 442 (2003) Wu, X.Y., et al., Nature Biotechnol., 21, 41 (2003) Jaiswal, J.K., H. Mattoussi, J.M.Mauro, S.M.Simon, Nature Biotechnol., 21, 47 (2003) Mansson, A. et al., Biochem. Biophys. Res. Commun., 314, 529 (2004) Torney, F. et al., Nature Nanotechnol., 2, (2007)

The present invention relates to a method for non-invasive delivery of sequence-specific nucleases into plant cells having cell walls using nanoparticles.

The following embodiments are described in conjunction with systems, tools and methods that are intended to be exemplary and exemplary rather than limiting in scope.

The present invention provides the following.
[1] A method for introducing a sequence-specific nuclease (SSN) into a plant cell,
Providing the plant cell having a cell wall;
The group consisting of electrostatic adsorption, conjugation to a ligand on the nanoparticle surface, coating of the nanoparticle with a small cofactor that SSN can recognize and bind, and direct conjugation to the nanoparticle Coating the surface of the nanoparticles with SSN by a method selected from:
Placing the cells having cell walls and the coated nanoparticles in contact with each other; and
Allowing the incorporation of the nanoparticles and the SSN into the plant cell comprising a cell wall.
[2] The method according to [1], wherein the step of coating the nanoparticles with SSN includes immobilizing the SSN on the surface of the nanoparticles by non-covalent absorption.
[3] The method according to [1], further comprising the step of absorbing the SSN into the nanoparticles.
[4] The method according to [1], further comprising the step of incorporating the nanoparticles into the plant cell compartment including a cell wall.
[5] The method according to [4] above, further comprising the step of coating the nanoparticles with a cell-penetrating peptide and / or an intracellular compartment targeting protein.
[6] The group consisting of cytosol, nucleus, tonoplast, plastid, ethioplast, chromoplast, white body, elaioplast, proteinoplast, amyloplast, chloroplast, and bilamellar lumen The method according to [4] above, which is selected from the above.
[7] The plant cell containing a cell wall is tobacco, carrot, corn, canola, rapeseed, cotton, palm, peanut, soybean, rice genus species (Oryza sp.), Arabidopsis sp. (Ricinus sp.) And the method according to [1] above, which is selected from the group consisting of sugar cane cells.
[8] In the above [1], the plant cell is derived from a tissue selected from the group consisting of embryo, meristem, callus, pollen, leaf, bud, root, root tip, flower, seed, pod and stem. The method described.
[9] The nanoparticles are gold nanoparticles, gold-coated nanoparticles, porous nanoparticles, mesoporous nanoparticles, silica nanoparticles, polymer nanoparticles, tungsten nanoparticles, gelatin nanoparticles, nanoshells, nanocores The method according to [1] above, selected from the group consisting of: nanospheres, nanorods, magnetic nanoparticles, semiconductor nanoparticles, quantum dots, nanomatrices, dendrimer nanomatrices, and combinations thereof.
[10] The method according to [1] above, further comprising derivatizing the surface of the nanoparticles.
[11] The method according to [1] above, wherein the SSN is a zinc finger nuclease composed of a zinc finger protein having a sequence-independent nuclease domain.
[12] The method according to [11] above, wherein the sequence-independent nuclease domain is derived from a type IIS restriction endonuclease FokI.
[13] The method according to [11] above, further comprising selecting a cell stably incorporating the ZFN.
[14] The method described in [13] above, wherein the selected cell is a reproducible cell.
[15] The method according to [14] above, further comprising the step of regenerating a plant from the renewable cell.
[16] The method according to [1] above, wherein the nanoparticles are multifunctional nanoparticles.
According to the present invention, a method for introducing a sequence-specific nuclease into a plant cell, comprising providing a plant cell having a cell wall, coating a nanoparticle with at least a sequence-specific nuclease, a plant cell having a cell wall, and Placing the sequence-specific nuclease-coated nanoparticles in contact with each other and allowing the uptake of the sequence-specific nuclease-coated nanoparticles into plant cells including the cell wall. Provided.

In addition to the exemplary aspects and embodiments described above, further aspects and embodiments include
It will become clear in view of the following description.

E. coli expression (1) of histidine tagged ZFN-IL1 Fok1. E. coli expression (2) of histidine untagged ZFN-IL1 Fok1. Figure 2 shows homologous recombination between chromosomes stimulated by IL-1 zinc finger-Fok1 fusion protein. A represents the target vector and B represents the recombinant with the rearranged GFP gene. A schematic representation of the plasmid pDAB1585 is shown. Shown are BY2-E single cell lines showing GNP-mediated YFP internalization 2 hours after cell incubation; Panel A (FITC), Panel B (Rhodamine), Panel C (DIC), Panel D (A + B), Panel E (A + B + C), Panel F (inverted reflectance image): YFP internalization as observed by fluorescence microscopy. Shows homologous recombination between chromosomes stimulated by the meganuclease I-SceI protein; A represents the target vector and B represents the recombinant with the rearranged GFP gene. A schematic representation of the plasmid pDAB100375 is shown.

In the description and tables below, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided:

Backcross. Backcrossing can be a process by which a breeder repeatedly crosses a hybrid seed progeny to one of its parents (eg, a process of crossing a first generation hybrid F ₁ with _one of the parental genotypes of the F ₁ hybrid). .

  Embryo. An embryo can be a small plant contained within a mature seed.

Nanoparticles. Fine particles having at least one nanoscale size, typically less than 100 nm. Nanoparticles suitable for use in the present invention can have a size of 1 nm to 0.4 um. The quantum dots can have a median diameter of 1 nm to 10 nm, preferably a median diameter of 2 nm to 4 nm. Nanoparticles as used in this application include gold nanoparticles, tungsten nanoparticles, gold-coated nanoparticles, porous nanoparticles, mesoporous nanoparticles, silica nanoparticles, polymer nanoparticles, gelatin nanoparticles, nanoshells , Nanocore, nanosphere
including, but not limited to, nanorods, magnetic nanoparticles, semiconductor nanoparticles, quantum dots, nanomatrices, dendrimer nanomatrices, and combinations thereof.

Quantum dots. Quantum dots are semiconductor nanoparticles that restrict the movement of conduction band electrons, valence band holes, or excitons (a constrained pair of conduction band electrons and valence band holes) in all three spatial directions. is there. This limitation is due to electrostatic potential (which is caused by external electrodes, doping, strain, impurities), the presence of interfaces between different semiconductor materials (eg in core-shell nanocrystal systems), the presence of semiconductor surfaces (Eg, semiconductor nanocrystals) or a combination thereof. A quantum dot can have a discontinuous quantized energy spectrum. The corresponding wave function is spatially localized inside the quantum dot, but extends beyond many periods of the crystal lattice. Quantum dots contain a small finite number of conduction band electrons (on the order of 1 to 100), valence band holes or excitons (ie, a finite number of elementary charges).

Nanomatrices include, but are not limited to, dendrimers. Dendrimers are spheroid or spherical nanoparticles that are either encased in their internal void space or manipulated to retain molecules that bind to the surface. Molecules are repetitively branched molecules; branching allows multivalent interactions between the surface and the bulk material. An example of a dendrimer is a spherical cationic polyamidoamine (PAMAM) cascade polymer. These polymers consist of primary amines on the surface and tertiary amines on the inside. This type of dendrimer is partially decomposed by heat treatment in a solvolytic solvent, thereby resulting in smaller steric constraints and greater flexibility. The large positive charge of the dendrimer facilitates electrostatic interaction with DNA, and its flexible structure allows the dendrimer to pack tightly when bound to DNA, and DN
Allow to swell when released from A. Transfection or transformation efficiency is increased as a result of the positive charge and flexible structural properties of the dendrimer. A variety of dendrimers can be obtained from Qiagen (Qiagen, Germany, MD), and such dendrimers are marketed as Superfect ™ transfection reagents (Cat # 301305).

Multifunctionalized. Unless otherwise specified, the term “multifunctionalized” is used to describe either monofunctionalized or multifunctional nanoparticles. Mono-functionalized nanoparticles are functionalized nanoparticles with only one type of functional group chemically attached, or only one type of functional group is chemically attached It shall mean an aggregate of nanoparticles. Multifunctionalized particles are at least two different types of functional groups, perhaps 3
It is intended to refer to a nanoparticle or nanoparticle aggregate in which two or more different types of functional groups are chemically bonded.

Resistant to herbicides. Resistance to a dose of herbicide inhibits growth,
And / or the ability of a plant to survive at that dose of an active ingredient that is believed to inhibit the survival of the non-resistant plant as a result (ie, the plant cannot be killed).
In some cases, resistant plants may temporarily turn yellow, or otherwise show some herbicide-induced injury (eg, excessive tillering and / or growth inhibition) Recover.

Stabilized. Stabilized refers to the characteristics of a plant that are reproducibly transmitted from one generation of inbred plants of the same variety to the next generation.

Capture. Incorporation can be a particle or matrix (eg, nanoparticles, etc., eg, gold,
Dendrimers or quantum dots) translocation across the cell wall or cell membrane, where the translocation is only as a result of the momentum imparted to the particle by something other than the cell in which the particle is being taken up It doesn't happen. Non-limiting examples of devices or methods that produce translocation of particles across the cell wall or cell membrane only as a result of the momentum imparted to the particles include biolistic technology, gene gun technology, microinjection technology and / or impalefection. ) Technology.

Nucleic acid. The terms “nucleic acid”, “polynucleotide” and “oligonucleotide” are used interchangeably and are in a linear or circular conformation and are either in single-stranded or double-stranded form. Deoxyribonucleotide polymer, ribonucleotide polymer, or
Other nucleotide polymers or nucleoside polymers are shown. For the purposes of this disclosure, these terms should not be construed as limiting with respect to the length of the polymer. These terms can include known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and / or phosphate moieties (eg, phosphorothioate backbones). In general, analogs of individual nucleotides have the same base pairing specificity; ie, analogs of A base pair with T.

Chromosome. A chromosome is a chromatin complex that constitutes the whole or part of a cell's genome.
A cell's genome is often characterized by its karyotype, which is a collection of all the chromosomes that make up the cell's genome. A cell's genome may contain one or more chromosomes. "
An “episome” is a replicating nucleic acid, nucleoprotein complex, or other structure that includes a nucleic acid that is not part of the chromosomal karyotype of a cell. Examples of episomes include plasmids and certain viral genomes. An “accessible region” is a site in cellular chromatin where a target site present in a nucleic acid can be constrained by an exogenous molecule that recognizes the target site. Although not wishing to be bound by any particular theory, accessible regions are considered to be regions that are not packed into the nucleosome structure. The unambiguous structure of the accessible region can often be detected by its sensitivity to chemical and enzyme probes (eg, nucleases). A “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule binds if sufficient conditions for binding exist. For example,
The sequence of 5 "-GAATTC-3 'is the target site for EcoRI restriction endonuclease.

gene. A gene is, for the purposes of this disclosure, a DNA region that encodes a gene product, and any that regulates the production of the gene product, regardless of whether the regulatory sequence is adjacent to the coding and / or transcriptional sequence. Of the DNA region. Thus, a gene is a promoter sequence,
Terminator, translational regulatory sequence (eg, ribosome binding site and internal ribosome entry site), enhancer, silencer, insulator, boundary element, origin of replication,
Including, but not necessarily limited to, matrix attachment sites and locus control regions.

Expression. The terms expression or gene expression are used interchangeably and indicate the conversion of information contained in a gene into a gene product. A gene product is a direct transcript of a gene (eg, mRNA, tRNA, rRNA, antisense RNA, ribozyme, structure R
NA or any other type of RNA) or protein produced by translation of mRNA. Gene products also include, for example, RNA modified by processes such as capping, polyadenylation, methylation and editing, and for example, methylation, acetylation, phosphorylation, ubiquitination, ADP ribosylation, myristylation and glycosylation. Proteins that are modified by chelation are included. “Modulation” of gene expression refers to a change in the activity of a gene. Expression regulation can include, but is not limited to, gene activation and gene repression.

protein. The terms “polypeptide”, “peptide” and “protein” are used interchangeably to denote a polymer of amino acid residues. This term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids.

An array. The term “sequence” refers to a nucleotide sequence of any length, which is DNA or RNA
It may be linear, cyclic or branched, and may be either single-stranded or double-stranded. The term “donor sequence” refers to a nucleotide sequence that is inserted into the genome. The donor sequence can be of any length: for example, 2 to 25,000 in length
Between 0 nucleotides (or any integer value between or above), preferably between about 100 and 5,000 nucleotides in length (or any integer in between) ), More preferably between about 200 and 2,500 nucleotides in length.

Homologous sequence. A homologous sequence refers to a first sequence that has some degree of sequence identity with a second sequence and that may be identical to the sequence of the second sequence. A “homologous non-identical sequence” is a first sequence that has some degree of sequence identity with a second sequence, but whose sequence is not identical to the sequence of the second sequence. . For example, a polynucleotide comprising the wild type sequence of the mutant gene is homologous and non-identical to the sequence of the mutant gene.
In certain embodiments, the degree of homology between two sequences is sufficient to allow for homologous recombination between them using normal cellular machinery. Two homologous non-identical sequences may be of any length, and the degree of their non-homology is (eg,
It may be as small as just one nucleotide (to correct genomic point mutations by targeted homologous recombination) or (eg, to insert a gene at a given site in a chromosome) It may be as large as 10 kilobases. Two polynucleotides comprising homologous non-identical sequences need not be the same length. For example, an exogenous polynucleotide of 20 to 10,000 nucleotides or nucleotide pairs (ie, a donor polynucleotide) can be used.

Recombinant. Recombination refers to the process of exchanging genetic information between two polynucleotides. For purposes of this disclosure, “homologous recombination (HR)” refers to a specialized form of such exchange that occurs, for example, during repair of double-strand breaks in cells. This process requires nucleotide sequence homology and uses a “donor” molecule for template repair of a “target” molecule (ie, a molecule that has undergone double-strand breaks), so that the process Cause the transfer of genetic information from target to target,
Variously known as “ver) gene conversion” or “short tract gene conversion”. While not wishing to be bound by any particular theory, such migration may be due to mismatch correction of heteroduplex DNA that occurs between the cleaved target and the donor, and / or if the donor is part of the target. May involve “synthesis-dependent strand annealing” (SDS) and / or related processes used to resynthesize the resulting genetic information. Such specialized HR often causes a change in the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

Cutting. “Cleavage”, “inducing a double strand break” and “cut” are used interchangeably to indicate that the covalent backbone of the DNA molecule is broken. . Cleavage is performed by enzymatic or chemical hydrolysis of phosphodiester bonds
Can be initiated by various methods, including but not limited to these. Both single-strand breaks and double-strand breaks are possible, and double-strand breaks can occur as a result of two separate single-strand break events. Cleavage of DNA can result in the production of either blunt ends or sticky ends. In certain embodiments, the fusion polypeptide is used for targeted double-stranded DNA cleavage. A “cleavage domain” includes one or more polypeptide sequences that have catalytic activity for DNA cleavage. The cleavage domain may be contained in a single polypeptide chain or the cleavage activity may result from the association of two (or more) polypeptides. A “cleavage half-domain” is a poly that forms a complex with a cleavage activity (preferably a double-strand cleavage activity) in cooperation with a second polypeptide (either the same or different). Peptide sequence. Double strand break and double strand break (doubl)
e stranded cleavage) is used interchangeably.

Chromatin. Chromatin is a nucleoprotein structure that constitutes the genome of a cell. Cellular chromatin includes nucleic acids, primarily DNA, and proteins (including histones and chromosomal proteins other than histones). The majority of eukaryotic cellular chromatin is present in the form of nucleosomes, where the core of the nucleosome is histone H2A, H2B, H3 and H4.
Each of which contains approximately 150 base pairs of DNA associated with an octamer, and linker DNA (of varying lengths depending on the organism) extends between the nucleosome cores.
Histone HZ molecules are generally associated with linker DNA. For the purposes of this disclosure, the term “chromatin” is intended to encompass all types of cell nucleoprotein, ie, both prokaryotic and eukaryotic cell nucleoproteins. Cellular chromatin includes both chromosomal and episomal chromatin.

Join. Binding refers to sequence specific non-covalent interactions between macromolecules (eg, between proteins and nucleic acids). As long as the overall interaction is sequence specific, not all components of the binding interaction need be sequence specific (eg, contact with phosphate residues in the DNA backbone). Such interactions are generally characterized by a dissociation constant (K _d ) of 10 ⁻⁶ M ⁻¹ or less. “Affinity” indicates the strength of binding: increased binding affinity correlates with lower K _d .

Operatable linkage. The terms “operable linkage” and the term “operably linked” (or the term “operably linked”) are interchangeable with respect to juxtaposition of two or more components (eg, sequence elements, etc.). In this case, in the juxtaposition of two or more components, these components are functions in which both components function normally and at least one of the components is exerted on at least one of the other components. It is arranged to allow the possibility of mediating. By way of example, if a transcriptional regulatory sequence controls the level of transcription of a coding sequence in response to the presence or absence of one or more transcriptional regulatory factors, a transcriptional regulatory sequence (eg, a promoter, etc.) Operatively linked. A transcriptional regulatory sequence is generally operably linked with respect to a coding sequence, but need not be directly adjacent to a coding sequence. For example, an enhancer is a transcriptional regulatory sequence that is operably linked to a coding sequence, even if the enhancer and coding sequence are not contiguous. With respect to fusion polypeptides, the term “operably linked” means that each of the components performs the same function in linking to other components that would perform if each of the components were not so linked. Can show the fact that For example, with respect to a fusion polypeptide in which a ZFP DNA binding domain is fused to a cleavage domain, in the fusion polypeptide, a ZFP DNA binding domain protein can bind to its target site and / or its binding site while cleavage. If the domain is capable of cleaving DNA in the vicinity of the target site, the ZFP DNA binding domain and the cleavage domain are in operable linkage.

Sequence specific nuclease (SSN) —this includes specific and unique nucleotide sequences (
Some classes of bifunctional proteins that can recognize native recognition sites or customized recognition sites include, but are not limited to, meganucleases, leucine zippers and zinc finger proteins. Meganuclease is
A group of enzymes capable of cleaving double-stranded DNA with high specificity in the presence of divalent metal ions (Ca, Mn, Mg). However, meganucleases differ from restriction endonucleases in their recognition properties and structure (Belfort, M., Roberts RJ, Homing endonucleases
: keeping the house in order, Nucleic Acid Research, 1997, 25: 3379-3388). Specifically, restriction enzymes recognize short nucleic acid sequences (3 bp to 8 bp), whereas meganucleases recognize longer sequences (12 bp to 40 bp), which improves DSB targeting. (Mueller, JE, Bryk, M., Loizos, N., Belfort M.,
Homing endonuleases, Nucleases (2nd edition), Linn, SM, Lloyd, RS, Roberts, RJ (edition), Cold Spring
Harbor Laboratory Press: 1993, 111-143). Leucine zippers are a group of proteins involved in protein-protein interactions in many eukaryotic regulatory proteins that are important transcription factors associated with gene expression. Leucine zippers display a common structural motif that is shared together in these transcription factors across several biological kingdoms, including animals, plants, yeast, and the like. Leucine zippers are attached to specific DNA sequences in such a way that the leucine residues are evenly spaced through the α-helix so that the leucine residues of the two polypeptides are on the same face of the helix. Formed by two polypeptides (homodimer or heterodimer) that bind.

Zinc finger DNA binding protein. A zinc finger DNA binding protein, ie, ZFP (or binding domain), is via one or more zinc fingers whose structure is a region of the amino acid sequence within the binding domain that is stabilized by the coordination of zinc ions. A protein that binds to DNA in a sequence-specific manner, or such a domain within a larger protein. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. A zinc finger binding domain can be “engineered” to bind to a predetermined nucleotide sequence. A non-limiting example of a method for manipulating zinc finger proteins is design and selection. Designed zinc finger proteins are non-naturally occurring proteins whose design / composition arises primarily from rational criteria. Rational criteria for design include the application of substitution rules, and the application of computer processing algorithms to process information in a database that stores information on existing ZFP designs and combined data. For example,
U.S. Patent Nos. 6,140,081, 6,453,242, 6,534,261
No. and 6,785,613. Similarly, International Publication Nos. 98153058, 98153059, 98153060, 021016536 and 031016496, and U.S. Pat. Nos. 6,746,838, 6,866,
See also 997 and 7,030,215.

Genome sequence. Genomic sequences include genomic sequences present on chromosomes, episomes, organelle genomes (eg mitochondria, chloroplasts), artificial chromosomes, and any other type of nucleic acid present on cells (eg duplex of amplified sequences). Micro sequences (amplified sequences double m
inute chromosomes), and endogenous and infected bacterial and viral genomes). Genomic sequences can be normal (ie, wild type) or mutated; mutated sequences can include, for example, insertions, deletions, translocations, rearrangements, and / or point mutations. The genomic sequence can also include one of many different alleles.

Plant cells. Plant cells include, but are not limited to, monocotyledonous plants (monocotyledonous plants) or dicotyledonous plants (dicotyledonous plants) or algae or moss cells. Non-limiting examples of monocotyledons include cereal plants such as corn, rice, barley, oat, wheat, sorghum, rye, sugarcane, pineapple, onion, banana and coconut. Non-limiting examples of dicotyledonous plants include tobacco, tomatoes, sunflowers,
Includes cotton, sugar beet, potato, lettuce, melon, soybean, canola (rapeseed) and alfalfa. Plant cells can be in any part of the plant and / or
It can be derived from any stage of plant development.

The target area. A region of interest is any region of a nucleic acid polymer that is desired to bind to an exogenous molecule (eg, a gene, a non-coding sequence within a gene, or a non-coding sequence adjacent to a gene, etc.). The binding can be for targeted DNA cleavage and / or targeted recombination. The target region is, for example, a chromosome, episome,
It may be present in an organelle genome (eg mitochondrion, chloroplast), plasmid, infectious virus genome or any other nucleotide sequence. The region of interest is non-coding within the coding region of the gene, inside a transcribed non-coding region (eg, leader sequence, trailer sequence or intron), or either upstream or downstream of the coding region. It can exist inside the transcription region. The region of interest may be as small as a single nucleotide pair in length, or up to 25,000 nucleotide pairs,
That is, it can be any integer value of nucleotide pairs.

According to an embodiment of the invention, a method for introducing a sequence-specific nuclease into a plant cell comprising a cell wall, wherein the sequence-specific nuclease-coated nanoparticles are placed in contact with the plant cell; and A method can be provided that includes allowing uptake across a plant cell wall. In a specific embodiment of the present invention, the nanoparticle may be any nanoparticle, the nanoparticle may contain zinc finger nuclease and / or meganuclease reversibly or irreversibly, zinc finger Can be coated with nuclease and / or meganuclease or otherwise bind to zinc finger nuclease and / or meganuclease and / or retain zinc finger nuclease and / or meganuclease can do. In certain embodiments, a zinc finger nuclease can be introduced into a nanoparticle prior to contact with a plant cell having a cell wall, or simultaneously with the introduction of the nanoparticle into a plant cell having a cell wall. Can be introduced. Examples of nanoparticles that can be used in embodiments of the present invention include gold, quantum dots, gold-coated nanoparticles, porous nanoparticles, mesoporous nanoparticles, silica nanoparticles, polymer nanoparticles, tungsten nanoparticles, gelatin nanoparticles. Particle, nanoshell, nanocore, nanosphere, nanorod, magnetic nanoparticle, semiconductor nanoparticle,
This includes, but is not limited to, quantum dots, nanomatrices, dendrimers and / or combinations thereof.

According to the embodiment of the present invention, the plant cell having a cell wall may be any plant cell as long as it is an intact and complete cell wall. Embodiments of the present invention include, but are not limited to, embryos, meristem cells, callus, pollen, leaves, cocoons, roots, root tips, flowers, seeds, pods, stems, suspension cultures and tissue cultures. It can include cells, including cell walls, from any tissue or any location where they are found.

In certain embodiments of the invention, the SSN can be any ZFN that can be delivered to plant cells according to the invention. For example, ZFN includes a fusion protein comprising a cleavage domain (or cleavage half-domain) and a zinc finger binding domain, polynucleotides encoding these proteins, and combinations of polypeptides and polynucleotides encoding polypeptides. obtain. A zinc finger binding domain may comprise one or more zinc fingers (eg, 2, 3, 4, 5, 6, 7, 8, 9, or more zinc fingers) Can also
It can be manipulated to join any desired area. Accordingly, a cleavage domain that is engineered to recognize a target sequence in said region of interest according to the methods disclosed herein by identifying the target region of interest for which cleavage or recombination is desired. One or more fusion proteins comprising (or cleaved half domains) and a zinc finger domain can be constructed. The presence of such a fusion protein (or protein) in the cell results in binding of the fusion protein to its binding site and cleavage within or near the region of interest. In addition, if an exogenous polynucleotide homologous to the region of interest is also present in such a cell, homologous recombination can be performed between the double-strand break nucleotide sequence and the exogenous polynucleotide. At a large rate.

In certain embodiments, providing at least one SSN to a cell can include providing one copy or more of the SSN protein directly to the cell via a nanoparticle. In other embodiments, providing the cell with at least one SSN provides the cell with:
Providing a nanoparticle comprising a nucleic acid encoding SSN and allowing a cell to produce SSN from a nucleic acid encoding SSN.

In other embodiments, the one or more SSNs provided to the cell are of interest 1
Cutting can be performed individually or in conjunction with other SSNs at or near one or more regions. In certain embodiments, one or more regions of interest can be expressed as a high expression protein, a higher expression protein, a very high expression protein, or
It can be present within the coding sequence of the most highly expressed protein. In some embodiments, the region or regions of interest is a highly expressed protein, a highly expressed protein, a very highly expressed protein, or a nucleotide sequence encoding the most highly expressed protein. It can be present near and / or within the containing locus. In other embodiments, the nucleotide sequence can be a double-strand break in only one region of interest. In a further embodiment, the nucleotide sequence can be a double strand break in more than one region of interest. In certain embodiments, one or more double-strand breaks are placed in the coding sequence of a highly expressed protein, a highly expressed protein, a very highly expressed protein, or the most highly expressed protein. be able to. In other embodiments, the one or more double-strand breaks comprise a nucleotide sequence encoding a highly expressed protein, a highly expressed protein, a very highly expressed protein, or a most highly expressed protein. It can be present near and / or within the locus.

In certain embodiments in which at least two double-strand breaks are made, repairing the double-strand break removes material during the double-strand break and rejoins both ends of the nucleotide sequence, resulting in two It may include excising sequences during single strand breaks. In embodiments, the excised sequence may be, without limitation, a high expression protein, a higher expression protein, a very high expression protein, or all or part of a nucleotide sequence encoding the highest expression protein. Can be included. In further embodiments, the excised sequence includes, without limitation, a high expression protein, a higher expression protein, a very high expression protein, or a regulatory sequence that achieves expression of the highest expression protein. obtain. In such embodiments, the expression of a high expression protein, a higher expression protein, a very high expression protein, or the highest expression protein is reduced compared to the level of expression prior to cleavage.

In an alternative embodiment in which at least two double-strand breaks are made, repairing the double-strand break removes material during the double-strand break and replaces it with a donor sequence, resulting in two Substituting the sequence during strand breaks with a donor sequence may be included. In other embodiments, the excluded sequence includes, but is not limited to, a high expression protein, a higher expression protein, a very high expression protein, or all or one of the nucleotide sequences encoding the highest expression protein. It may contain a sequence encoding part. In further embodiments, the excluded sequences can include, but are not limited to, high expression proteins, higher expression proteins, very high expression proteins, or regulatory sequences that achieve expression of the highest expression proteins. . In such embodiments, the expression of a high expression protein, a higher expression protein, a very high expression protein, or the highest expression protein is reduced compared to the level of expression prior to cleavage.

In an alternative embodiment in which a single double strand break is made, repairing the double strand break is performed by placing the donor sequence into the double strand break or across the double strand break.
break) may include inserting. In certain embodiments, the donor sequence can be inserted into a highly expressed protein, a higher expressed protein, a very high expressed protein, or the coding sequence of the highest expressed protein. In embodiments, the insertion of such a sequence includes, as a non-limiting example, a high expression protein, a higher expression protein, a very high expression protein due to the presence of an in-frame stop codon. Alternatively, transcription of the coding sequence of the most highly expressed protein can be interrupted. In further embodiments, the donor is, but is not limited to, a high expression protein, a higher expression protein,
It can interfere with the function of regulatory sequences to achieve expression of very highly expressed proteins or the most highly expressed proteins. In various embodiments, the expression of a high expression protein, a higher expression protein, a very high expression protein, or the highest expression protein is
Reduced compared to the level of expression prior to cleavage.

In still other embodiments, the donor sequence can encode a protein of interest. In further embodiments, expression of the protein of interest from the donor sequence can be controlled by regulatory sequences present in the donor sequence and / or regulatory sequences present in the sequence into which the donor sequence is inserted, or It can be regulated by such regulatory sequences or can be operably linked to such regulatory sequences. In a further embodiment, the nucleic acid sequence encoding the protein of interest is separate from the donor sequence,
Alternatively, it can be provided to cells in conjunction with a donor sequence. In some embodiments,
The donor sequence can be contained in the same nucleic acid molecule as the sequence encoding the protein of interest.

In other embodiments, a highly expressed protein, a highly expressed protein, a very highly expressed protein, or a nucleotide sequence of a highly expressed protein encoding the most highly expressed protein, a nucleotide of a highly expressed protein Non-limiting examples of sequences, nucleotide sequences of highly expressed proteins, or nucleotide sequences that encode the nucleotide sequences of the most highly expressed proteins include genomes, plasmids, cosmids, artificial chromosomes, episomes, or others in cells In the nucleotide structure.

Implementation of the above methods, as well as the preparation and use of the compositions disclosed herein, unless otherwise indicated, such as molecular biology, biochemistry, chromatin, which are within the skill of the art. Conventional techniques in structure and chromatin analysis, computational chemistry, cell culture, recombinant DNA and related fields are used. These techniques are explained fully in the literature. For example, Sambro
ok et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition, Cold Spring Harbor Laboratory
Press, 1989, and 3rd edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
John Wiley & Sons, New York, 1987 and regular revisions; METHODS IN ENZYMOLOGY series, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, 3rd edition, Academi
c Press, San Diego, 1998; METHODS IN ENZYMOLOGY, 304, `` Chromatin '' (PMWassarman
And APWolffe), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLO
See GY, Vol. 119, “Chromatin Protocols” (PBBecker), Humana Press, Totowa, 1999.

Nucleic Acid Delivery to Plant Cells As noted above, DNA constructs can be introduced into a desired plant host (eg, its genome) by a variety of conventional techniques. For a review of such technologies,
For example, Weissbach & Weissbach, Methods for Plant Molecular Biology (1988, Academic Pr
ess, NY), section VIII, pages 421-463, and Grierson & Corey, Plant Molecular Biol.
See ogy (1988, 2nd edition), Blackie, London, Chapters 7-9.

For example, the DNA construct can be introduced directly into the genomic DNA of the plant cell using various techniques, such as, for example, electroporation and microinjection into plant cell protoplasts, or the DNA construct Can be introduced directly into plant tissue using biolistic methods, such as using DNA particle bombardment (see, eg, Klein et al. (1987) Nature, 327: 70-73. ).
Alternatively, the DNA construct can be combined with a suitable T-DNA flanking region and introduced into a conventional Agrobacterium tumefaciens host vector. Agrobacterium tumefaciens-mediated transfection techniques are well described in the scientific literature, including the disarming and use of binary vectors. For example, Horsch et al. (1984), Science, 233: 496-498, and Fraley et al. (1983), Proc
Nat. Acad. Sci. USA, 80: 4803.

In addition, gene transfer can be accomplished using non-Agrobacterium bacteria or viruses, such as Rhizobium sp. NGR234, Sinorhizoboium meliloti, Mesorhizobium roti (Mesorhizobium roti)
loti), potato X virus, cauliflower mosaic virus and cassava vein mosaic virus and / or tobacco mosaic virus. See, for example, Chung et al. (2006), Trends Plant Sci., 11 (1): 1-4.

In addition, cell penetrating peptides fused to nanoparticles or sequence specific nucleases can be used to deliver nucleotide or protein sequences into plant cells. Nanoparticles for expressing and isolating cell penetrating peptides for delivery in plant cells,
It can be functionalized with nucleotide sequences or proteins. Cell penetrating peptides that can functionally deliver various molecules into plant cells are known in the art, and include TAT (Chugh et al. (2008), FEBS, 275: 2403-2414), R9. (Chang et al. (2005), Plant Cel
l Physiol, 46 (3): 482-488 and Chen et al. (2007), FEBS Lett, 581 (9): 1891-1897), MP
G (Ziegler et al. (2008), Adv Drug Deliver Rev, 6: 580-597 and Morris et al. (1997), Nuclei
c Acids Res., 25: 2730-2736), PEP1 (Henriques et al. (2005), Biochemistry-US, 44 (3):
10189-10198) and plant-derived cell penetrating peptides, but are not limited to these.

The virulence function of the Agrobacterium tumefaciens host is that the cells can be expressed in binary T-DNA vectors (Bevan (1984), Nuc. Acid Res., 12: 8711-8721) or co-culture techniques (Ho
rsch et al. (1985), Science, 227: 1229-1231) leads to the insertion of constructs and flanking markers into the DNA of plant cells when infected by this bacterium. Generally, Agrobacterium transfection systems are used to manipulate dicotyledonous plants (Bevan et al. (1982), Ann. Rev. Genet, 16: 357-384; Rogers et al. (1986), Methods Enz.
ymol., 118: 627-641). Agrobacterium transfection systems can also be used to transform monocotyledonous plants and plant cells, as well as to introduce DNA into monocotyledonous plants and plant cells. U.S. Patent No. 5,591,616; Hemalsteen et al. (1984), EMBO J, 3: 3039-3041; Hooykass-Van Slogteren et al. (1984), Nature, 3
11: 763-764; Grimsley et al. (1987), Nature, 325: 1677-179; Boulton et al. (1989), Plant Mol. Bio
l., 12: 31-40; and Gould et al. (1991), Plant Physiol., 95: 426-434.

Alternative gene transfer and transfection methods include protoplast transfection by calcium mediated uptake, polyethylene glycol (PEG) mediated uptake or electroporation mediated uptake of naked DNA (Paszkowski et al. (198
4), EMBO J, 3: 2717-2722; Potrykus et al. (1985), Molec. Gen. Genet., 199: 169-177; From et al. (1
985), Proc. Nat. Acad. Sci. USA, 82: 5824-5828; and Shimamoto (1989), Na
ture, 338: 274-276) and electroporation of plant tissue (D'Hall
uin et al. (1992), Plant Cell, 4: 1495-1505). Additional methods for transfection of plant cells include microinjection, silicon carbide-mediated DNA uptake (Kaeppler et al. (1990), Plant Cell Reporter, 9: 415-418), and microprojectile bombardment (Klein et al. (1988), Proc. Nat. Acad. Sci.
USA, 85: 4305-4309; and Gordon-Kim et al. (1990), Plant Cell, 2: 603-618)
Is included.

The disclosed methods and compositions can be used to insert exogenous sequences at predetermined locations in the plant cell genome. This is useful because the expression of the transgene introduced into the plant genome is highly dependent on its integration site. Thus, for example, a gene encoding a nutrient, antibiotic or therapeutic molecule can be inserted into a region of the plant genome that is advantageous for its expression by targeted recombination.

Transfected plant cells produced by any of the above transfection techniques can be cultured to regenerate a complete plant having the transfected genotype and thus the desired phenotype. it can. Such regeneration techniques rely on the manipulation of several plant hormones in tissue culture growth media, typically biocide markers and / or herbicides that have been introduced along with the desired nucleotide sequence. Rely on drug markers. Plant regeneration from cultured protoplasts is described by Evans et al., “Protoplas
ts Isolation and Culture ", Handbook of Plant Cell Culture (pages 124-176, Macmilli
an Publishing Company, New York, 1983) and Binding, Regeneration of Plants, Plant
Protoplasts (pages 21-73, CRC Press, Boca Raton, 1985). Regeneration can also be obtained from plant callus, explants, organs, pollen, embryos or parts thereof. Such regeneration techniques are generally described in Klee et al. (1987), Ann. Rev. of Plant Phys., 38: 467-486.

Nucleic acids introduced into plant cells can be used to confer essentially any desired trait to any plant. A wide variety of plant and plant cell systems can be used to produce the desired physiological and crop agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transfection methods described above. Can be operated for. In preferred embodiments, target plants and plant cells for manipulation include such monocotyledonous and dicotyledonous plants, such as cereal crops (eg, wheat, corn, rice, millet, barley), fruit crops. (E.g. tomatoes, apples, pears, strawberries, oranges), feed crops (e.g. alfalfa), root crops (e.g. carrots, potatoes, sugar beets, yams), leafy crops (e.g. lettuce, spinach, cabbage) Including crops;
Flowering plants (eg petunia, roses, chrysanthemum), conifers and pine (eg pine, fir,
Spruce); plants used in phytoremediation (eg, heavy metal-accumulating plants);
Oil crops (eg, sunflower, rapeseed, tides, palms) and plants used for experimental purposes (eg, Arabidopsis), etc. are included but are not limited to these. Accordingly, the disclosed methods and compositions are provided by Asparagus, Avena.
Brassica, Citrus, Watermelon (Citrullus), Capsicum, Pumpkin (Cucurbita), Carrot (Daucus), Soybean (Glycine
), Cotton genus (Gossypium), barley genus (Hordeum), chinus genus (Lactuca), tomato genus (Lycopersicon), apple genus (Malus), cassava (Manihot), tobacco genus (Nico)
tiana), rice genus (Oryza), crocodile genus (Persea), pea genus (Pisum), pear genus (Pyr)
us), cherry (Prunus), radish (Raphanus), rye (Secale), eggplant (
A wide variety of species, including but not limited to species from Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea Used across plants.

One skilled in the art will recognize that after an expression cassette is stably incorporated into a transgenic plant and confirmed to be operable, the expression cassette can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used depending on the species to be crossed.

Identifying and isolating transfected plant cells, callus, tissue or plants by selecting or screening for engineered plant material for traits encoded by marker genes present in the DNA for transfection Can do.
For example, the selection can be made by growing plant material that has been engineered in a medium containing an inhibitory amount of an antibiotic or herbicide to which the genetic construct for transfection confers resistance. In addition, transfected plants and plant cells are also identified by screening for the activity of any visible marker gene (eg, β-glucuronidase gene, luciferase gene, B gene or C1 gene) that may be present in the recombinant nucleic acid construct. can do. Such selection and screening methodologies are well known to those skilled in the art.

Physical and biochemical methods can also be used to identify plant transfectants or plant cell transfectants that contain the inserted gene construct. These methods include: 1) Southern analysis or PCR amplification to detect recombinant DNA insert and determine its structure, 2) Northern blot, primer extension to detect and examine RNA transcripts of gene constructs PCR amplification or reverse transcriptase PCR amplification, 3) enzyme assay to detect enzyme activity or ribozyme activity (when such gene product is encoded by a gene construct), 4) protein gel electrophoresis, Western blot Techniques, immunoprecipitation or enzyme-linked immunoassays (when the product of the gene construct is a protein), 5) single nucleotide polymorphism detection techniques, invader assays, pyrosequencing, solexa sequencing However, it is not limited to these. Additional techniques such as in situ hybridization,
Enzymatic staining and immunostaining can also be used to detect the presence or expression of a recombinant construct in a particular plant organ or plant tissue. Methods for performing all these assays are well known to those skilled in the art.

The effects of genetic manipulation using the methods disclosed herein can be observed, for example, by Northern blots of RNA (eg, mRNA) isolated from the tissue of interest. Typically, if the amount of mRNA is increased, it can be assumed that its corresponding endogenous gene is being expressed at a greater rate than before. Other methods of measuring gene activity can be used. Different types of enzyme assays, the substrate used, and
It can be used depending on the method of detecting the increase or decrease of the product or by-product of the reaction. In addition, the level of expressed protein is determined immunochemically, ie, E well known to those skilled in the art.
It can be measured by LISA, RIA, EIA, and other assays based on antibodies, such as by electrophoretic detection assays (with either staining or Western blotting). The transgene can be selectively expressed in several tissues of the plant or at several developmental stages, or the transgene can be substantially all along its entire life cycle. Can be expressed in plant tissues. However, any combinatorial expression pattern is also applicable.

The present disclosure also encompasses the seeds of the transgenic plants described above, where
The seed has a transgene or gene construct. The disclosure further encompasses a progeny, clone, cell line or cell of the transgenic plant described above, wherein the progeny, clone, cell line or cell has a transgene or gene construct.

Applications The disclosed methods and compositions for targeted cleavage can be used to induce mutations in genomic sequences. Targeted cleavage can also be used to create gene knockouts or gene knockdowns (eg, functional genomics or target validation)
It can also be used to facilitate targeted insertion (ie, sequence knock-in) of sequences into the genome. Insertion is, as a non-limiting example, via homologous recombination,
Alternatively, it may be by replacement of the chromosomal sequence by targeted integration where a new sequence (ie, a sequence that is not present in the region of interest) is inserted into a given target site. In certain instances, such a new sequence can be flanked by sequences homologous to the region of interest on the chromosome. The same method can be used to replace a wild-type sequence with a mutated sequence, or to change one allele to a different allele.

Treating a pathogenic infection in a plant host by targeted cleavage of an infectious plant pathogen or an integrated plant pathogen, for example, by cleaving the genome of the pathogen such that its pathogenicity is reduced or eliminated Can be used for. In addition, targeted cleavage of genes encoding receptors for plant viruses prevents the expression of such receptors,
Thereby, it can be used to prevent viral infection and / or viral spread in the plant.

Typical plant pathogens include various plant viruses such as Alfalnovirus (Alfa
rnovirus), Alphacryptovirus, Badnavirus, Betacryptovirus, Bigigeminivirus, Bromovirus, Bymovirus, Capirovirus, Carlavirus, Carrnovirus, Carimovirus, Closterovirus, comovirus, Cucurnovirus, cytolabed virus, dianthosovirus, enamovirus, fabavirus, physivirus, furovirus, Foldivirus (H
ordeivirus), Hybrigeminivirues, Idaeovirus, Ilawirus, Ipomovirus, Luteo virus, Macromovirus, Macluravirus, Malafivirus, Monogeminivirues , Nanaviruses, Necrovirus, Nepovirus, Nucleovirus virus, Oriza virus, Urmiavirus, Phytoreovirus, Potexvirus, Potyvirus, Rymovirus, Satellite WAS , Satellite virus,
Sekivirus, Sobemovirus, Tenui virus, Tobamovirus, Tobra virus, Tornbus virus, Tospovirus, Toriko virus, Timovirus, Umbravirus, Varicosavi virus
rus) and Waika viruses; fungal pathogens such as smut fungi (eg, Ustilaginales), rust fungi (Uredinales), ergots (Clavicepts pu)
purea) and powdery mildew fungi; mold (Oomycetes), such as phytofutra
Phytophthora infestam (potato wilt), etc .; bacterial pathogens, such as the genus Erwinia (eg, E. herbicola), Pseudomonas (Pseudom)
onas) (eg, P. aeruginosa, P. syringae, P. fluorescense, and P. putida)
, Ralstonia genus (eg R. solanacearum), Agrobacterium (Ag
robacterium and Xanthomonas genus; roundworm (Nemato
da)); and, but not limited to, Phytomyxea (Polymyxa and Plasmodiophora).

The disclosed method for targeted recombinant production of a protein of interest can be used to replace any genomic sequence with a non-identical sequence. For example, a mutant genomic sequence can be replaced by its wild-type counterpart, thereby providing a method for treating plant diseases, conferring resistance to plant pathogens, and reducing crop yield It can be increased. In a similar manner, one allele of a gene can be replaced by a different allele using the targeted recombination methods disclosed herein.

In many of these cases, the region of interest contains a mutation and the donor polynucleotide contains its corresponding wild type sequence. Similarly, wild type genomic sequences can be replaced by mutant sequences if such is desired. For example, overexpression of an oncogene is reversed, either by mutating the gene or by replacing its regulatory sequences with sequences that support lower non-pathological levels of expression. Can do. In fact, any pathology that depends on a particular genomic sequence, in any manner, can be cured or reduced using the methods and compositions disclosed herein.

Targeted cleavage, insertion, excision and / or recombination can also be performed on non-coding sequences (eg,
Regulatory sequences (eg, promoters, enhancers, initiators, terminators, splice sites, etc.) can be altered and used to alter the level of expression of the gene product. Such methods can be used, for example, for therapeutic purposes, functional genomics and / or target validation studies.

Targeted modifications of the chromatin structure can be used to facilitate binding of the fusion protein to cellular chromatin. In a further embodiment, a 1 between the zinc finger binding domain and the recombinase (or functional fragment thereof)
One or more fusions may be used in addition to or instead of the zinc finger-cleavage domain fusions disclosed herein to facilitate targeted recombination. it can. See, for example, shared US Pat. No. 6,534,261 and Akopian et al. (20
03), Proc. Natl. Acad. Sci. USA, 100: 8688-8691. In further embodiments, the disclosed methods and compositions comprise a transcriptional activation domain or transcriptional repression that requires a fusion of a ZFP binding domain for its activity, either homodimerization or heterodimerization. Used to give a domain. In these cases, the fusion polypeptide comprises a zinc finger binding domain and a functional domain monomer (eg, a monomer derived from a dimeric transcription activation domain or transcription repression domain). Two such fusion polypeptides can be dimerized by binding to an appropriately placed target site so that a functional transcriptional activation or repression domain is reconstituted. become.

Furthermore, as disclosed above, the methods and compositions presented herein can be used for targeted integration of exogenous sequences into a region of interest in the genome of a cell. In this case, for example, by cleavage, insertion by a homology-dependent mechanism (eg either identical to a given genomic sequence (ie target site) or homologous but non-identical) The insertion of donor sequences comprising exogenous sequences together with one or more sequences that are

The donor sequence supports homology-directed repair of double-strand breaks in the genomic sequence, thereby allowing the exogenous sequence to be inserted at the genomic target site in a region adjacent to the exogenous sequence,
It can contain sufficient homology. Thus, the donor nucleic acid has a homology-dependent repair mechanism (
It can be of any size sufficient to support the integration of exogenous sequences (eg, by homologous recombination). Although not wishing to be bound by any particular theory, the homologous regions located on both sides of the exogenous sequence are located at the ends of the cleaved chromosomes with templates for the resynthesis of genetic information at the site of double-strand breaks. It is possible to provide. In certain embodiments, two of the same sequence or two of the homologous but not identical sequences (or one of each) are present adjacent to the exogenous sequence. An exogenous sequence (or exogenous nucleic acid or exogenous polynucleotide) is one that contains a nucleotide sequence that is not normally present in the region of interest.

Exemplary exogenous sequences include, but are not limited to, cDNA, promoter sequences, enhancer sequences, epitope tags, marker genes, cleavage enzyme recognition sites, and various types of expression constructs. See, for example, US Pat. No. 6,833,252. Further representative homing endonucleases include I-CeuI, PI-Ps
pI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII,
I-PpoI, I-SceIII, ICreI, I-TevI, I-TevII and I-
Tai III is included. Their recognition sequences are known. US Pat. No. 5,420,03
2; Belfort et al. (1997), Nucleic Acids Res., 25: 3379-3388; Dujon et al. (1989), Gene, 82: 115.
-118; Perler et al. (1994), Nucleic Acids Res., 22, 1125-1127; Jasin (1996), Trends Genet.,
12: 224-228; Gimble et al. (1996), J. Mol. Biol., 263: 163-180; Argast et al. (1998), J. Mol. Bio
l., 280: 345-353 and see also New England Biolabs catalog.

Marker genes include sequences encoding proteins that mediate antibiotic resistance (eg, ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), colored proteins or fluorescent proteins or photoproteins (eg, green Sequences encoding fluorescent proteins, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins that mediate enhanced cell proliferation and / or gene amplification (eg,
Including, but not limited to, sequences encoding (dihydrofolate reductase). Therefore, representative marker genes include β-glucuronidase (GUS), phosphinothricin N-acetyltransferase (PAT, BAR), neomycin phosphotransferase, p-lactamase, catechol dioxygenase, a-amylase, tyrosinase, P- Galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR)
Including, but not limited to, dalapon dehalogenase and anthranilate synthase. In some embodiments, targeted integration is used to insert sequences involved in the regulated expression of RNA expression constructs, eg, microRNA or siRNA. As described above, promoters, enhancers and additional transcriptional regulatory sequences can also be incorporated into the RNA expression construct.

In conventional transformations, random integration of foreign DNA is used to create modified transgenic crop plants with selected traits that are severely limited in some foreign markets. In addition, undesired results also arise from the method of DNA transfer or from random insertion of transgenes (often multiple times per genome) into vulnerable regions of the genome. In particular, the effects of inaccurate insertion may not appear in early generations. This is because various DNA error blocking mechanisms are activated during growth, reproduction, embryogenesis and development. These results affect the time and cost of any transgenic program in agricultural biotechnology. However, the recent invention of Dow AgroSciences (International Publication No. 2008/2008/0212)
07)), a method for the correct insertion of the transgene is described by zinc finger nuclease (ZFN) mediated homologous recombination. Conversely, if the ZFN protein can be expressed, purified outside the target organism, and then delivered to the target plant cell, a surgically specific mutation / gene knockout can result in non-homologous end joining (NHEJ). ). Thus, the present invention can produce non-transgenic genetically modified plants that would avoid the limitations associated with transgenic crops, and that the targeted gene editing process requires a transgenic approach. It is possible.

Various methods are known in the art for heterologous expression of sequence-specific nuclease proteins (eg, ZFN proteins, etc.). Applicable expression systems include the use of in vitro systems, such as wheat germ cell-free systems (eg, US Pat. No. 7,235,38).
No. 2, which is incorporated herein by reference); Pseudomonas fluorescens expression system (Madduri et al. (2007), Protein Expres Purif, 55 (2): 352-360); and Pichia ) Protein expression system (US Pat. Nos. 4,683,293 and 4)
808,537, 4,812,405, 4,818,700, 4,
No. 837,148, No. 4,855,231, No. 4,857,467, No. 4,8
79,231, 4,882,279, 4,885,242, 4,89
5,800, 4,929,555, 5,002,876, 5,004
688, 5,032,516, 5,122,465, 5,135,
See 868, 5,166,329. These include, but are not limited to, uses such as those incorporated herein by reference.

Certain embodiments of the invention are conjugated to nanoparticles (NP) to induce double-strand breaks and restoration of the functionality of disrupted genes by NHEJ, and are NP-mediated, stealthy. A) Exogenously expressed functional ZFN delivered to intact plant cells by the delivery method. In other embodiments, the functional ZFN conjugated to NP is
Delivered with donor fragments of DNA by an NP-mediated, stealth delivery method. In that case, ZFN cuts a specific sequence in the genome and the donor DNA is integrated into this locus by homologous recombination. A strategy for linking proteins to NPs is:
Four major approaches have been taken: (1) electrostatic adsorption, (2) conjugation to ligands on the NP surface, (3) to small cofactor molecules that can be recognized and bound by proteins. Conjugation and (4) Conjugation directly to the NP surface (Aubin-Tam and Hamad-Schifferli, 2008). Other strategies are described in the review by Medintz et al. Problems involved in these labeling strategies include sterics or whether the protein can “beyond” the ligand and reach the NP surface or appropriate linking group. The selection of chemistry that results in specific linkage (ie, does not cause excessive crosslinking) and is stable for the desired purpose is also a necessary consideration. ZFN peptides need to be functionalized under high DDT concentrations and in the presence of zinc ions. In order to preserve the stability of the conjugate, functionalization is performed according to the conjugation procedure described in Oh et al. (2010).

  The invention is further described by the following illustrative examples.

Example 1: Production of sequence specific nuclease (SSN) by in vitro translation or bacterial expression

Manipulating SSNs (IL1-LO / Fok1, IL1-43 / Fok1, IL1-8 / Fok1 and I-SceI) and PCR amplification from plasmids containing SSN, thereby creating a restriction enzyme site and a 6 × histidine tag Add. The PCR product is inserted into TOPO vector pCR2.1 for cloning and sequencing. The gene fragment containing the SSN coding sequence is removed from the plasmid by restriction digestion and expressed in the expression vector pET1
Ligate to a compatible restriction site in 5b. These samples were mixed with pDAB4883 B
Transform into L21 expressing competent E. coli cells. A plasmid consisting of a pCOT4 expression plasmid containing a ligase gene downstream of a promoter that is similarly induced by IPTG (isopropyl-β-D-thiogalactopyranoside) to damage SSN protein for effective expression Reduce in BL21 E. coli DNA by transforming BL21 with pDAB4883. This helps to repair any damage caused by SSN to the BL21 cell genome during the overexpression period.

The ligase gene-pCOT4 construct and the SSN-pET15b construct are cotransformed into the same BL21 expressing cells. Transgenic BL21 cultures were grown in 50 mL of LB medium containing chloramphenicol, carbenicillin and ZnCl ₂ and OD _600n
Incubate at 37 ° C. until _m reaches 0.5. Expression at various concentrations of IPTG
(0.1 mM to 0.7 mM) and incubation is carried out at various temperatures (from 16 ° C.
28 ° C.) and analysis is performed by SDS-PAGE and Western blot to detect the presence of SSN protein. Thus, IL1-LO / Fok1, IL1-43
/ Fok1, IL1-8 / Fok1 and I-SceI are expressed in E. coli cells, and Ni-N
TA purified. After purification, the function of SSN is demonstrated based on the ability to release specific fragments from the expression plasmid.

Alternatively, the sequence specific nuclease is expressed by in vitro translation. Promega
The commercial kit TNT® obtained from a provides an efficient and convenient process for expressing proteins. T7 RNA polymerase promoter or SP6
A circular plasmid containing a sequence-specific nuclease gene cloned downstream of either of the RNA polymerase promoters is expressed in vitro by a protein expression enzyme supplied with the kit. Synthesized protein is produced in 50 μL reaction within 60-90 minutes according to manufacturer's protocol. Additional commercial kits are available for in vitro translation of proteins, and additional kits that can be used include, in addition to other commercially available kits, ActivePro ™, Ambion from Ambion PROTEINscript obtained from (
Trademark) II, PURExpress obtained from New England Biolabs
s (TM) is included.

Figures 1 and 2 show E. coli expression of histidine-tagged SSN (ZFN-IL1Fok1) (
1) and E. coli expression (2) of histidine untagged SSN (ZFN-IL1Fok1). In vitro expressed ZFN-IL1Fok1 releases a well-defined ZFN binding site flanking fragment from the plasmid. Thus, both E. coli expressed SSN and in vitro expressed SSN are useful for efficient and specific digestion of target DNA molecules and they are used alternately throughout this study.

The protein expressed from the cloned SSN gene is shown to be functional (ie, functional in cleaving donor DNA). For example, (
Plasmid pDAB1585 (shown in FIG. 4) is treated with ZFN-IL1Fok1. The digested plasmid DNA is linearized. The linearized fragment derived from the ZFN-IL1Fok1-digested plasmid is then purified from the gel and self-ligated using an overnight in vitro ligation procedure. Transfer the ligation product to chemically competent DH5α E. coli cells. Several recombinant colonies are recovered and analyzed by both restriction pattern analysis and DNA sequencing, which reveals that pDAB1585 digests as expected.

Example 2-SSN flanked by GFP reporter gene fragment
Production of target cell cultures with binding sites

Two green fluorescent protein (gfp) gene fragments (Evogen Joint Sto) whose target sequence is located next to the β-glucuronidase (uidA) expression cassette
ck Company, Moscow, Russia). In one target construct, a ZFN binding site (FIG. 3) with a recognition sequence consisting of inverted repeats that allow the zinc finger-Fok1 fusion protein to bind as a homodimer is incorporated into the target construct. The binding site contains four tandem repeats of the recognition sequence of the IL1-Fok1 fusion protein, so that each binding site is accessible to the zinc finger-Fok1 fusion protein in a chromatin environment where the recognition sequence is complex. To guarantee, the size is about 200 bp. In the second construct, an I-SceI binding site is incorporated into the target construct (FIG. 6). In each target construct, the binding site is fused with the uidA coding sequence at the N-terminus. The 5 'and 3' gfp gene fragments overlap by 540 bp.
These overlapping sequences provide homology within the target sequence, and the stop codon ensures that no functional gfp transcription from the target sequence takes place at the 3 ′ end of the 5 ′ gfp fragment. Inserted into.

Target sequences are stably incorporated into BY2 tobacco cell suspension cultures using Agrobacterium transformation. BY2 cultures (obtained from Nihon Tobacco (Iwata, Shizuoka, Japan) Uejun) at a pH of 6.0 LS basal medium (PhytoTechnology)
y Labs, Shawnee Mission, KS, # L689), 170 mg / L
KH ₂ PO ₄ , 30 g / L sucrose, 0.2 mg / L 2,4-D and 0.6 mg
Maintain in medium containing / L thiamine-HCL. BY2 cells were added to 0.25 mL of PC
Subculture every 7 days by adding V to 50 mL of LS-based medium and maintaining in a 250 mL flask on a rotary shaker at 25 ° C. and 125 RPM. To create a transgenic BY2 cell culture with an integrated target sequence, the suspension flask 4 days after subculture was divided into 10-12 4 mL aliquots, and these aliquots were PZFN-TARGET pDAB1 grown to OD ₆₀₀ of about 1.5 overnight
100 × 2 with 100 μL of Agrobacterium LBA4404 strain carrying either 585 (FIG. 4) or pI-SceI-TARGET pDAB100375 (FIG. 7)
Co-culture in 5 mm petri dishes. The dish is a Nescofilm® (A
zwell Inc. , Osaka, Japan) and incubated for 3 days at 25 ° C. without shaking, after which excess liquid is removed and replaced with 11 mL LS medium containing 500 mg / L carbenicillin. After resuspension of tobacco cells, 1 mL of suspension was added to 8 g / L TC agar (PhytoTechnology, Shawnee Missi).
on, KS) and dispensed into 100 × 25 mm plates of LS medium containing 500 mg / L carbenicillin and 200 mg / L hygromycin. Plates are incubated at 28 ° C. in the dark without wrapping. Thereby, 120-14
Four selection plates are provided for one treatment. Individual hygromycin resistant isolates appear 10-14 days after placement and are transferred to individual 60 × 20 mm plates (one isolate per plate). On plates, isolates are kept under selection as callus on a 14 day subculture schedule until needed for analysis and subsequent retransformation experiments.

A hygromycin resistant transgenic cell culture containing one copy of the target sequence incorporated in full length contains about 250 mg to 500 mg of callus tissue and 100 mg / L of hygromycin as described above. Select by placing in 40 mL to 50 mL LS basal medium and subculture every 7 days and use to restart suspension culture.

Cell clusters and single cells (these are single-cell patent applications filed by DAS (WO / 2/2)
008/083233) are used in the experiments.
From 3 days to 4 days before the experiment, the suspension culture after 1 week was changed to 2 mL of BY2 suspension aggregate (
Stock concentration of 4-chloro-1,5-diphenyl-1H-pyrazol-3-yloxy) acetic acid ethyl ester, as described in patent No. 2008/083233, 1% to 3%
Subculture into fresh medium by transferring glycerol and 0.05% -0.1% (v / v) DMSO to 40 mL LSBY2 medium contained in a 250 mL flask. Single cells are collected either 3.5 days or 7 days after treatment to induce single cells. BY
Two single cells are processed through a flow cytometer to determine cell viability and are also processed to assess cell stability by confocal microscopy. Stability is detected by observing the level of background fluorescence, if background fluorescence is present. A small percentage of cells show background fluorescence corresponding to dead cells, indicating that background fluorescence is from cells that have undergone necrosis. Both single cells and normal suspension clumps are used in experiments after testing for background fluorescence.

Example 3-Coating with SSN to deliver nanoparticles (NP) into plant cells Gold colloid with a diameter of 150 nm (BBI International, GC1)
50), 5-((2- (and-3) -S (acetylmercapto) succinoyl) amino)
Fluorescein (SAMSA fluorescein: Invitrogen, A-685), carboxylic acid polyfunctionalized gold colloidal nanoparticles with sizes of 80 nm and 90 nm (TedP
ella, 32019), sulfo-NHS (N-hydroxysulfosuccinimide), E
DC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) (Pi
erce Biotechnology, 24510, 22980), MES (2- [N
-Morpholino] ethanesulfonic acid) (Fisher Scientific, AC327)
76-1000), phosphate buffered saline buffer packet (Sigma, P5368-
10 PAK), histidine-tagged GFP (Evrogen, excitation max. -482 nm, emission max. -502 nm, FP611), turbo YFP (Evrogen, excitation max. -525 n)
m, emission maximum -538 nm, FP611), propidium iodide (Sigma, P48)
64), fluorescein diacetate (Sigma, F7378) is a different type of polyfunctionalized NP that is coated with SSN and used for delivery into target cell cultures.

(I) Preparation of nanoparticle conjugates (a) Synthesis of gold-fluorescein conjugates without SSN for control treatment: gold-fluorescein conjugates deliver particles in BY2 clusters or BY2 single cells without SSN To track and track previously described methods (Can
none et al., 2006). 100 mg of 1 mg SAMSA fluorescein
Dissolve in μl 0.1 M NaOH and vortex for 15 minutes to remove the acetyl group protecting the thiol. This activated SAMSA is then mixed with 100 μl of 150 nm gold colloid (about 109 particles / mL). This solution is then incubated for 1 hour to ensure completion of the reaction. Then 50 μL of 1M HCl is added to neutralize the solution. Centrifuge the solution at 3000 RPM for 30 minutes and remove the supernatant. The resulting yellow pellet is resuspended in 200 μL 0.1 M PBS, thereby obtaining an orange solution. This purification process is repeated twice to ensure the removal of free SAMSA fluorescein.
The manner in which SAMSA binds to gold is mainly through thiol bonds. Due to the significant electrostatic repulsion (SAMSA is dianionic at a pH above 7),
It is believed to lie perpendicular to the gold colloid surface (Cannone et al., 2006). Such NPs are used to track the entry of the number of such fluorescent emitting particles that enter the cell as a measure of cell compliance in a given condition.

(B) Synthesis of SSN-coated gold nanoparticles (GNP): GNP-SSN conjugates are synthesized using a slightly modified protocol described by Grabarek and Gergely (1990). 0.25 mL of a solution of carboxylic acid polyfunctionalized gold colloid from 20 nm to 150 nm (approximately 109 particles / mL) at 3000 RPM at 10
Centrifuge for minutes. After discarding the supernatant, the red pellet was reconstituted with 1 mL of activation buffer (0.1 M
Suspend in MES, 0.5M NaCl, pH 6.0). Then 0.4 mg EDC and 1.1 mg sulfo-NHS are added to the solution and vortexed for 15 minutes at room temperature. 9 μL of ZFN-IL1Fok1 is then added and the resulting solution is incubated at room temperature for up to 2 hours in the dark for complete reaction of protein and gold.
The ratio of gold colloid and protein used in this reaction is determined by finding the number of carboxylic acids present on the surface of the gold colloid. First, the number of carboxyl groups present on the surface of one gold colloid is determined by the surface area of one gold particle (assuming a sphere) (the surface occupied by one carboxyl group (0.20 nm ² (Kimura et al., 2002)). Calculate by dividing by)). This result is then multiplied by the total number of colloidal gold present to obtain the total number of carboxyl groups present throughout the colloidal gold solution. This is considered equivalent to the number of amino groups present in a given amount of protein. This gold colloid binds to the protein by the formation of an amide bond between the carboxylic acid present on the surface of the gold colloid and the amino group present on the surface of the protein (Grabarek and Gergely, 1990). Roughly 127,285 protein molecules are linked to one gold nanoparticle.

  (Ii) Cell treatment:

a) Time course of gold uptake and cell viability: Prepare the following samples in a 24-well sterile plate: (i) 500 μL of target suspension cluster or target single cell (control), (ii) 500 μL of BY2 suspension. Cluster or BY2 single cell + 20 μL of G
NP + 25 μL of fluorescein diacetate (FDA) +25 μL of propidium iodide, and (iii) other treatments, along with cells and cell viability dyes, 40 μL, 60
μL or 80 μL of GNP is included alone and in combination with ZFN-IL1Fok1. Treated samples are examined with a fluorescence microscope at 5 minutes, 20 minutes, 120 minutes and finally after 24 to 48 hours to confirm cell viability.

b) Gold-SAMSA fluorescein treatment: Prepare the following samples in a 24-well sterile plate before the experiment: (i) 500 μL target suspension cluster or target single cell (control), (ii) 500 μL target suspension cluster or Target single cell + 20 μL S
Treat AMSA-fluorescein (control) and (iii) target suspension cluster or target single cell + 20 μL GNP-SAMSA-fluorescein and allow suspension to remain in the dark for 20 minutes at room temperature to confirm particle ingress Incubate.

c) GNP-coated (eg tagged) ZFN-IL1Fok1 treatment—The following samples are prepared in 24-well sterile plates prior to cell treatment or suspension cluster treatment: (i
) 500 μL of target suspension cluster or target single cell (control), (ii) 500 μL
L target suspension cluster or target single cell + 9 μL-20 μL of ZFN (control),
And iii) 500 [mu] L of single cells + 10 [mu] L to 40 [mu] L of GNP-coated (e.g. tagged) ZFN-IL1Fok1. Treated cells and clusters are incubated in the dark at room temperature for up to 2 hours before the experiment.

All experiments to ensure that the control treatment using GFP / YFP-bound GNP ensures non-invasive penetration and the optimal timing of entry used as a guide in the experiment (see Figure 5) Included in In addition, a fused cell penetrating peptide (CPP) is fused (eg, polyfunctionalized) to the NP to track the real-time entry of particles into the target suspension cluster and target single cell.

Example 4: Synthesis of Quantum Dot (QD) Tagged SSN Conjugates QD of luminescent semiconductor nanocrystals provides a powerful prototype example with many proven biological applications (Thermes V et al., 2002 Windbichler et al., 2007; Fajardo-Sanchez et al., 200
8; Amould S et al., 2006). Their utility comes from a combination of unique photophysical characteristics and a size comparable to the size of large proteins. The hydrodynamic radius of hydrophilic CdSe-ZnS QD ranges from about 5 nm (for nanocrystal caps exchanged with molecular ligands) to about 20 nm (for nanocrystals encapsulated in block copolymers) (Smi).
th J et al., 2006). A single QD provides several biomolecules (eg, antibodies, peptides, DNAs) to provide increased avidity to the coated QD bioconjugate.
Etc.).

ZFN-IL1Fok1 conjugated (eg, coated) to hydrophilic QD
Are described in this example as an alternative strategy to facilitate their intracellular uptake and intracellular delivery to appropriate target DNA sites. This method is generally referred to as D
Follow the procedure for internalization of QD-protein cargo into living cells as described in AS patent application 65502.

(I) QD synthesis: CdSe-ZnS with emission maximum centers at 510 nm and 540 nm
Core-shell QD according to a procedure described for the stepwise reaction of organometallic precursors in a hot coordinating solvent mixture (Lu et al., 2007; Doyon et al., 2006; Collins et al., 2003; Lanio et al., 2000) Use to synthesize. The original capping shell, composed primarily of trioctylphosphine and trioctylphosphine oxide (TOP / TOPO), was prepared as previously described (Lie et al., 2002; Mami et al., 2005; Desjarlais And Berg, 1993), making it multifunctional and hydrophilic by exchanging with a bifunctional ligand. Two sets of hydrophilic QDs are used: (1) nanocrystals capped only by dihydrolipoic acid, and (2) poly (ethylene glycol) -added dihydrolipoic acid (PEG Mw≈600, DHLA-PEG) and Biotin-terminated DHLA-poly (ethylene glycol) (PEG Mw≈400, DHLA
-Nanocrystals capped with a mixture of (PEG-biotin) with a ligand molar ratio of 9: 1. These are denoted as DHLA-QD and DHLA-PEG-biotin-QD, respectively.

(Ii) Self-assembly of quantum dot bioconjugate: QD-ZFN-IL1Fok1
In order to self-assemble the conjugate with the desired valence, His-
ZFN was added to 0.3 nM, 510 n in 10 mM Tris-Cl (pH 8) buffer.
In addition to DHLA-capped QD emitting m, incubate for 30 minutes at room temperature.
Similarly, b-PE-streptavidin was added to phosphate buffered saline (137 mM).
NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4, PBS).
3 μM 540 nm emitting QD (which is capped with a 9: 1 molar ratio of DHLA-PEG: DHLA-PEG-biotin) plus incubation overnight at 4 ° C .; conjugate formation in this case Is caused by the streptavidin-biotin interaction. The conjugate is characterized using gel electrophoresis. In gel electrophoresis, changes in electrophoretic mobility of QDs assembled with either His-added ZFN or streptavidin labeled b-PE are monitored. Samples were washed with 1x TBE buffer (0
. 09M Tris, 0.002M Na2-EDTA, 0.09M boric acid, pH 8.3)
1% or 2 for QD-b-PE conjugate and YFP conjugate
Run each on a% agarose gel. In particular, if the ZFN-IL1Fok1 molecule is fused to a fluorescent protein for tracking, ZFN-I per QD bioconjugate
The effect of changing the number of L1Fok1 molecules is monitored. Gel images are collected by exciting QDs and / or proteins and capturing fluorescent images of separated bands in the gel. Conjugate formation is confirmed by monitoring changes in energy transfer between the QD and the fluorescent protein when self-assembled. Fluorescence spectra were obtained using Tecan Safari Dual Mon using 325 nm excitation.
ochromator Multifunctional Microtiter Plate Reader (Tecan, Re
search Triangle Park, NC). For intracellular delivery and imaging experiments, QDs are self-assembled with a mixture of ZFN-IL1Fok1 at a normal ZFN: QD molar ratio.

(Iii) Intracellular uptake of quantum dot-fluorescent protein conjugate: Cell internalization experiments are performed in aseptic conditions as previously described. The QD bioconjugate is diluted in complete culture medium and added to the cell culture and added at 40 μg / mL to 150 μg / mL 3
Incubate for 1 hour at 7 ° C. Along with CPP with 50 CPPs per QD,
1: 5 or 1:10 QD / ZFN and QD / with a collective valency of 1: 1 to 1: 2.5
Mixed surface QD-coated conjugates consisting of any of the b-PEs are incubated with cell cultures at various QDF conjugate concentrations. Excess unbound QD conjugate is removed by washing the culture at least three times with PBS or cell culture medium. Cells are then fixed in 3.7% paraformaldehyde for 10 minutes at room temperature, washed twice with PBS, and fixed in ProLong Antifade fixation medium containing DAPI dye (Invitrogen) for nuclear staining. Collection of epi-fluorescence images
Perform using a Leica microscope. Separate fluorescent images for each slide are collected and quantified using a DualView system equipped with a 565 nm dichroic filter. For cellular imaging of 510 nm emission QD-YFP / ZFN, the sample was excited at 488 nm and the emission was 5
Collect / separate with 65 nm dichroic filter and deconvolve. The fluorescence of QD is collected at λ less than 565 nm and the fluorescence tail of YFP is collected at λ greater than 565 nm. Q
The YFP leakage into the D region is subtracted as part of the deconvolution process. QD and b-PE with 540 nm radiation are excited at 488 nm and their respective emissions are separated by a 565 nm dichroic filter and deconvolved. The DAPI fluorescence is excited using a Xe lamp and the radiation is excited by the DAPI cube (D350 / 50 × for excitation, bicolor 40
0 DCLP, D460 / 50 m for detection). AF647-TF
Excitation is performed using a Xe lamp and fluorescence is detected using a Cy5 cube (excitation HQ620 / 50 ×, two-color Q660LP, emission HQ700 / 75 m). Both excitation cubes / detection cubes are provided by Chroma Technology. Differential interference contrast (DIC) images are collected using a bright light source.

(Iv) Confirmation of ZFN-IL1Fok1-coated QD: His interaction is nanocrystalline Zn
It occurs directly on rich inorganic surfaces. Two (His) separated by a small spacer
) ZFN-I with N-terminus having 6 sequences and CPP with N-terminal (His) 8 sequences
By manipulating L1Fok1, it is possible to form a strong QD-protein / peptide complex. Biotin-avidin binding is a bioconjugation strategy used everywhere known in the art because of its strong interaction (KD of about ^10-15 M). Q capped with a mixture of hydroxyl-terminated PEG and biotin-terminated PEG
By using the D surface (DHLA-PEG-biotin-QD) b
-Easy conjugation (eg coating) to PE-streptavidin is possible.

(V) Intracellular delivery of QD-ZFN-IL1Fok1 conjugate: YFP / ZFN-
To confirm that the incorporation of polyfunctionalized (eg, surface functionalized) QD coated by IL1Fok1 cargo is mediated by the presence of CPP at the nanocrystal surface
Incubate the target BY2 cell line separately with the three types of conjugates:
QD-CPP conjugate (10-100 CPPs per QD), QD-ZFN-
QD assembled with IL1Fok1 / CPP and a mixture of ZFN-IL1Fok1 and CPP (about 10 ZFN-IL1Fok1 and about 5 per conjugate)
QD-ZFN-IL1Fok1 / CPP with 0 CPP). Cells are incubated with a solution of QD conjugate emitting at 510 nm (at a concentration of about 75 nM), rinsed to remove any unbound, and then imaged using epifluorescence microscopy. The cells also have DAPI to allow nuclear and endosomal visualization respectively.
Is counterstained. When additional CPP is present on the QD surface (mixed surface QD-ZF
N-IL1Fok1-CPP conjugate), substantial intracellular uptake of the conjugate occurs as indicated by the significant fluorescence intensity measured for both sets of cells. Furthermore, the images collected for both cultures show that almost complete overlap is observed for QD and ZFN-I
It shows that it exists between the fluorescence patterns of L1Fok1 / YFP. Evaluation of staining patterns and co-localization patterns indicates that the distribution around the nucleus and is mainly confined to the endosomal compartment. Efficient internalization of QD conjugates by cell lines in the presence of CPP is indicated by CPP being multifunctionalized by the ZFN-IL1Fok1-fluorescent fusion protein cargo (
For example, it will be shown to facilitate intracellular uptake of QD that is surface functionalized.

Single cells and agglomerate clusters are treated similar to the NP treatment described in the nanoparticle section, placed without selection, and monitored for GFP expressing colonies 2-4 weeks after the experiment.

Example 5-Homology-directed repair after SSN delivery into tobacco cells by GNP and QD, as described above, integrated ZFN treated with ZFN-IL1Fok1 or I-SceI binding to particles A target cell line having a binding site or I-SceI binding site is placed in a medium in a Petri dish. Cells are placed in non-selective medium after treatment. The center of green fluorescence is observed after 7 days. To confirm that the observed fluorescence results from the reconstitution of the functional gfp gene, a group of fluorescent tissue segments is isolated and manually enriched by several selective subcultures. Genomic DNA is isolated from these fluorogenic tissues and analyzed by PCR using a probe that binds to any gfp gene fragment. Samples enriched from SSN-treated fluorogenic tissues, when amplified, yield the expected 0.6 kb PCR product, which, due to the expected recombination, is functional gfp gene. Shows that these organizations have been restructured. An additional 4.1 kb PCR product was also observed in the enriched sample, indicating the presence of a non-recombinant reporter gene in the cell population.
This is not unexpected when considering the method of macroscopic selection of fluorogenic tissues used to achieve enrichment of gfp positive cells.

Numerous exemplary aspects and embodiments have been discussed above, and those skilled in the art will recognize some modifications, permutations, additions and subcombinations thereof. Accordingly, the following appended claims and any claims subsequently introduced include all such modifications, permutations, additions and subcombinations as fall within the true spirit and scope thereof. It is intended to be interpreted as follows.

Claims (14)

A method for introducing a sequence-specific nuclease (SSN) into a plant cell comprising:
Coating the nanoparticles with SSN, wherein the nanoparticles are gold nanoparticles or gold-coated nanoparticles;
Placing the cells having cell walls and the coated nanoparticles in contact with each other; and allowing the uptake of the nanoparticles and the SSN into the plant cells comprising the cell walls. .   The method of claim 1, further comprising providing a plant cell having a cell wall first.   3. The method of claim 2, wherein the step of coating the nanoparticles with SSN comprises immobilizing the SSN on the surface of the nanoparticles by non-covalent absorption.   3. The method of claim 2, further comprising the step of incorporating the nanoparticles into the plant cell compartment comprising a cell wall. The method according to any one of claims 1 to 4 , further comprising a step of coating the nanoparticles with an intracellular compartment targeting protein. The compartment is selected from the group consisting of cytosol, nucleus, tonoplast, plastid, ethioplast, chromoplast, white body, elaioplast, proteinoplast, amyloplast, chloroplast, and bilamellar lumen. The method according to claim 4 . The plant cells including cell walls are tobacco, carrot, corn, canola, rapeseed, cotton, peanut, soybean, Oryza sp., Arabidopsis sp., Ricinus sp. The method according to any one of claims 1 to 6 , wherein the method is derived from a plant selected from the group consisting of sugarcane cells. The plant cell, embryo, meristematic, callus, derived from pollen, leaf, anthers, roots, root tips, flowers, seeds, tissue selected from the group consisting of sheath and stem, claim 1-7 The method according to one item.   The method of claim 2, further comprising derivatizing a surface of the nanoparticles.   The method according to claim 2, wherein the SSN is a zinc finger nuclease (ZFN) composed of a zinc finger protein having a sequence-independent nuclease domain, preferably derived from the type IIS restriction endonuclease FokI. 11. The method of claim 10 , further comprising selecting cells that stably incorporate the ZFN. The method of claim 11 , wherein the selected cell is a regenerative cell. 12. The method of claim 11 , further comprising regenerating a plant from the renewable cell. The nanoparticles are multifunctional nanoparticles, the method according to any one of claims 1 to 13.

Images